Natural Killer (NK) immune cells are essential to human health, performing key roles in surveillance and killing of cancerous and infected cells. Activated NK cells harness and deliver lytic granule-contained, cytotoxic proteins through a specialized area of cell-cell contact (immunological synapse) onto the offending cell. Over the past decade, our laboratory has greatly advanced the understanding of how NK cells use the precisely regulated steps constituting directed secretion to achieve cytotoxic function and control. In this program renewal, we build on our past discoveries to ask critical questions at the root of NK cell biology. In Aim 1, we ask why multiple pathways to degranulation exist and whether these can be exploited to match specific clinical scenarios. In the previous program, we reported that lytic granules converge to the microtubule organizing center (MTOC) prior to secretion. We hypothesize that convergence focuses an NK cell's killing effect on a single diseased cell (minimizing collateral damage to healthy cells), whereas dispersion promotes wider destruction in environments full of targets. Here, we test these hypotheses using cutting-edge, high- and super- resolution microscopy in conjunction with the only immunologically-dedicated ultrasound guided acoustic trap microscopy (UGATm) cell manipulation system available in the U.S. We will determine whether receptor blocking and/or inhibitors can be used to fine tune convergence to match specific disease contexts like extracellular pathogen infection or advanced cancer. In Aim 2, we define a new cooperative motility mechanism for degranulation. In the previous program, we discovered that granules are dynamic even after delivery to the synapse, and that degranulation occurs through a nanoscale mesh of actin pores. Here, we define the dynamics and movement mechanisms of both synaptic granules and the actin meshwork using advanced super-resolution microscopy. We hypothesize that these two independent systems utilize dynamics to promote pore finding, efficient degranulation, and ultimately cytotoxicity. This dual-dynamic paradigm would be new to cell biology and would set the stage for therapeutic strategies to enhance NK cell killing by promoting granule access to the synapse. Finally, in Aim 3, we investigate a new role for Vimentin and the intermediate filament (IF) cytoskeleton in the establishment of synapse polarity. Vimentin IF, like synaptic actin, granules, and the MTOC, are dynamic, but intriguingly, they show reverse polarity, localizing to the opposite side of the NK cell. Here, using high-throughput imaging flow cytometry, super-resolution microscopy, inhibitors, and gene targeting, we will test the hypotheses that IF respond to activating signals, establish the distal NK cell pole, and are critical for synapse polarity. This will be the first demonstration of IF as a means to cell polariy. Based on our success in the previous program, we expect the proposed experiments to lead to key insights into directed secretion for NK cell cytotoxicity. We further expect the paradigms that emerge to have broad biological significance and direct applicability to immunological and cell-based therapies.